Project Summary/Abstract A common feature of human aging is an increased susceptibility to infections. This age-related susceptibility is linked with a significant and reproducible loss of nave T cells in older individuals. Notably, aged nave cells not only reduce in number but also display features of partial differentiation, implying a loss in cellular quiescence may cause repertoire contraction and dysfunction with age. Nave T cell quiescence is classically maintained within secondary lymphoid tissues (SLTs) by fibroblastic reticular cells (FRCs). Mouse models have elegantly demonstrated the ability of FRCs to promote T cell survival and directly inhibit activation-induced differentiation. These data suggest that FRCs actively regulate nave T cell quiescence and likely play an essential role in its loss during aging. However, mechanistic studies in humans are significantly confounded by rare use of single cell technologies and limited by poor nave T cell survival in standard in vitro culture and humanized mice. Better utilization of new single cell techniques and the development of novel methodologies that sustain nave T cells in vitro is thus required to gain further insight into nave T cell maintenance during human aging. Our preliminary data suggests high heterogeneity of human nave T cells that narrows with age, a situation that can be mimicked in vitro through the use of a novel SLT-like organoid model system. For this proposal, Aim 1 will define the overall phenotypic, transcriptional and epigenetic heterogeneity of the nave T cell compartment during human aging. In addition, Aim 2 will further develop SLT-like organoids for the long- term study of nave T cell quiescence. Aim 3 will use our organoid system, in combination with ex vivo and in vitro assays, to determine the role of FRCs in the maintenance of nave T cell quiescence and its breakdown with aging. The overarching goals of these studies are to define fundamental nave T cell heterogeneity and determine causes of age-related homeostatic decline ? to ultimately identify potential therapeutic interventions to boost protective immunity and prevent pathogenic infections in older individuals. The applicant Dr. Claire Gustafson has outlined an integrative five-year research and career development plan to advance her knowledge in single cell techniques and their analysis pipelines and to develop a novel, organoid-based research platform to study T cell homeostasis during aging. Dr. Gustafson?s mentor, Dr. Jrg Goronzy, has comprehensive expertise in T cell aging as well as an extensive network of scientific collaborators using high throughput analytical techniques. This mentorship combined with Dr. Gustafson?s team of expert advisors and the resources available at Stanford University provides an exceptional research environment for Dr. Gustafson to successfully become a strong independent investigator in the area of human immune aging.